Antibiotic contamination of milk and related dairy products is a significant problem in the dairy industry. A major source of such contamination are betalactam antibiotics (e.g., penicillins and cephalosporins), which have been administered to cows to treat mastitis or other infections. More generally, large quantities of milk and related dairy products are discarded each year due to betalactam antibiotic contamination, thereby causing substantial economic losses.
There are several known procedures for detecting betalactam antibiotics in a liquid sample, known procedures such as microbiological and chemical techniques, high-performance liquid chromatography, and enzyme immunoassays. For example, microbiological (agar diffusion) techniques permit detection of about 0.1 to 0.5 microgram of antibiotic per milliliter of sample (i.e., .mu.g/ml), but such techniques are rather time-consuming (see, e.g., Bennett et al., "Simplified Accurate Method For Antibiotic Assay of Clinical Specimens," Appl. Microbiol. 14:170-177, 1966; Cole et al., "Metabolism of Penicillins to Penicilloic Acids and 6-APA in Man and its Significance in Assessing Penicillin Absorption," Antimicrob. Agents Chemother. 3:463-468, 1973; and Spyker et al., "Pharmacokinetics of Amoxicillin: Dose Dependence After Intravenous, Oral and Intramuscular Administration," Antimicrob. Agents Chemother. 11:132-141, 1977). Chemical procedures are generally somewhat faster, but are typically much less sensitive (see, e.g., Marelli, L. P., "Analytical Procedures For Cephalosporins" in E. H. Flynn (ed.), Cephalosporins and Penicillins, Academic Press, Inc., New York, 1972, pp. 609-635). High-performance liquid chromatography has been used to estimate amoxycillin and ampicillin in sera and urines (see, e.g., Vree et al., "Rapid Determination of Amoxycillin (clamoxyl) and Ampicillin (penbritin) in Body Fluids of Many By Means of High Performance Liquid Chromatography," J. Chromatogr. 145:496-501, 1978) and cephalosporin C in fermentation media (see, e.g., Alemanni et al., "HPLC Routine Analysis of Biosynthetic Active Compounds in Fermentation Media," Chromatographia 12:396-398, 1979); in these chromatographic techniques, however, minimal concentrations of about 0.5 .mu.g/ml are generally necessary. Finally, an enzyme immunoassay has been devised which detects ampicillin at concentrations as low as 10 ng/ml; betalactam antibiotics other than ampicillin, however, have not been investigated by this technique (see, e.g., Kitagawa et al., "Novel Enzyme Immunoassay of Three Antibiotics: New Methods For Preparation of Antisera to the Antibiotics and for Enzyme Labeling Using a Combination of Two Hetero-Bisfunctional Reagents," S. B. Pai (ed.), Enzyme Labelled Immunoassay of Hormones and Drugs, Walter de Gruyter, Inc., Hawthorne, N.Y., 1978, pp. 59-66).
In addition to these known procedures, several commercial methods have been developed for detecting betalactam antibiotics in milk. For example, U.S. Pat. Nos. 4,239,745 and 4,239,852 to Charm describe commercial methods for detecting an antibiotic in a liquid sample (e.g., milk) based on the competitive binding between the antibiotic contaminant and a tagged antibiotic to receptor sites on bacterial cells. These commercial assays, like some of the other proposed assays, are based on immunochemical reactions and utilize antibodies directed against specific betalactam antibiotics. There are, however, several disadvantages associated with these types of assays, including: (1) the sample generally requires removal of interfering materials; and (2) the sample additives generally require a mixture of antibodies with specificities and high affinities for different betalactam antibiotics.
Of the commercial methods developed for detecting the presence of betalactam antibiotics, the enzymatic methods based on the ability of betalactam antibiotics to inactivate a specific D,D-carboxypeptidase bacterium Actinomadura-R39 are of particular interest (see, e.g., Frere et al., "Enzymatic Method for Rapid and Sensitive Determination of .beta.-Lactam Antibiotics," Antimicrobial Agents and Chemotherapy, p. 506-510, 1980). Although other bacterial D,D-carboxypeptidase are known to be reversibly inhibited by betalactam antibiotics, the R39 enzyme has been preferentially used because the rate of inactivation is very rapid and the reversal of inhibition is very slow. Thus, over short periods of time, exposure of R39 enzyme to a betalactam antibiotic results in a stoichiometric loss of R39 catalytic activity. Accordingly, measurement of remaining R39 activity after exposure to test samples (suspected of containing a betalactam antibiotic) provides an assay for detecting the antibiotic.
More specifically, the assay as described by Frere et al. is similar to a commercial test known as PENZYM.TM. sold by UCB Bioproducts (Brussels, Belgium). This commercial test, however, is rather time-consuming and involves a number of steps and separate reagents. The first step involves an incubation (e.g., five minutes) of the test sample with the carboxypeptidase. If the test sample contains a betalactam antibiotic, a certain amount of the enzyme will be inactivated during the incubation depending on the amount of antibiotic present. The next two steps involve adding a substrate for the carboxypeptidase, which is a peptide containing a carboxyterminal D-alanine-D-alanine. This is followed by another incubation (e.g., 15 minutes) during which the terminal D-alanine is cleaved from the substrate. Other reagents are added during this incubation period to measure the amount of cleaved D-alanine. The liberated D-alanine is oxidized into pyruvic acid by a D-amino acid oxidase enzyme with simultaneous formation of hydrogen peroxide. The hydrogen peroxide oxidizes an organic redox indicator (e.g., o-dianisidine), which provides a colorimetric read-out. Sulfuric acid is added at the end of the incubation period to terminate the reaction and stabilize the color formation.
The PENZYM.TM. kit is supplied with seven separate reagents including: (1) the D,D-carboxypeptidase; (2) buffer for the D,D-carboxypeptidase; (3) substrate for the D,D-carboxypeptidase ((Acetyl).sub.2 -L-Lys-D-ala-D-ala); (4) flavin adenine dinuclcotide, cofactor of the D-amino acid oxidase; (5) peroxidase; (6) o-dianisidine and (7) D-amino acid oxidase. The use of this kit, however, suffers from a number of disadvantages. First, the sequential addition of reagents in several different steps is required. Second, the amount of time required to complete the assay (i.e., about 20 to 30 minutes) is considered excessive, particularly by milk haulers. Finally, an excessive number of separately-packaged reagents must be handled.
Accordingly, there is a need in the art for improved methods for detecting betalactam-containing compounds, particularly in the context of detecting the presence of a betalactam antibiotic in a liquid sample. The present invention fulfills these needs, and provides further related advantages.